Disposable bioreactor for culturing microorganisms and cells

Abstract

A disposable bioreactor for culturing microorganisms and cells is provided. The bioreactor is suitable for use by individuals not skilled in microbiology or aseptic technique. It is constructed of flexible or semi-flexible waterproof sheets to form a container designed to provided mixing and gas exchange to microorganisms cultured therein. Mixing and gas exchanged are achieved by bubbling gas through the culture, either from a single locus at the lowermost apex of a container having a wedge-shaped or rounded bottom, or from multiple loci across a flat-bottomed container. Also provided is a kit for culturing a selected microorganism, preferably an entomopathogenic nematode or other organism useful as a biopesticide.

We claim:  
     
       1. A disposable bioreactor for culturing microorganisms or cells, comprising flexible or semi-flexible waterproof sheets sealed along their edges to form a container, the container having a wedge shaped or rounded bottom, at the lowermost extremity of which is positioned a gas bubbler for generating gas bubbles for mixing and providing gases to microbial or cellular liquid cultures within the container, the container further comprising at least one inlet port for introducing gases or liquids and at least one exit port for exhausting gases or removing liquids wherein the exit port further comprises a coiled exhaust tube extending therefrom. 
     
     
       2. The disposable bioreactor of  claim 1 , wherein the container is generally square to rectangular except for the bottom, with a length to width ratio of about 1:1 to about 10:1. 
     
     
       3. The disposable bioreactor of  claim 2 , wherein the length to width ratio of the container is between about 2:1 and 5:1. 
     
     
       4. The disposable bioreactor of  claim 1 , wherein the container is two-sided. 
     
     
       5. The disposable bioreactor of  claim 1 , wherein the container is at least three-sided. 
     
     
       6. The disposable bioreactor of  claim 1 , wherein the container further comprises a partition extending longitudinally from the center of the container in either direction partially to the top and bottom, such that liquid in the container can flow over or under the partition. 
     
     
       7. The disposable bioreactor of  claim 6 , wherein the gas bubbler is positioned asymmetrically to one side of the partition. 
     
     
       8. The disposable bioreactor of  claim 1 , wherein the gas bubbler comprises a gas inlet port through which gas is pumped. 
     
     
       9. The disposable bioreactor of  claim 8 , wherein the gas inlet port is connected by a gas inlet tube to a gas pump. 
     
     
       10. The disposable bioreactor of  claim 9 , wherein the gas inlet tube comprises at least one filter for filtering microbial contaminants. 
     
     
       11. The disposable bioreactor of  claim 8 , wherein the gas inlet port is located at the lowermost extremity of the container. 
     
     
       12. The disposable bioreactor of  claim 8 , wherein the gas inlet port is located at the top or on a side of the container, and further comprises a tube internal to the container, which extends and is affixed to or near the lowermost extremity of the container. 
     
     
       13. The disposable bioreactor of  claim 1 , wherein the gas bubbler further comprises a gas diffuser for breaking large gas bubbles into smaller gas bubbles. 
     
     
       14. The disposable bioreactor of  claim 1 , wherein the exhaust tube is between about 3 inches and about 5 feet in length. 
     
     
       15. The disposable bioreactor of  claim 1 , wherein the exhaust tube further comprises a valve to prevent back flushing of liquids to the interior of the container. 
     
     
       16. The disposable bioreactor of  claim 1 , wherein the exhaust tube further comprises a filter for filtering microbial contaminants. 
     
     
       17. The disposable bioreactor of  claim 1 , which further comprises an inoculation port for introducing inoculafit into the container. 
     
     
       18. The disposable bioreactor of  claim 17 , wherein the inoculation port comprises a septum. 
     
     
       19. The disposable bioreactor of  claim 1 , which further comprises a breakable pouch on an interior face of the container. 
     
     
       20. The disposable bioreactor of  claim 1 , which further comprises an external reservoir in fluid communication with the container interior. 
     
     
       21. A kit for culturing a microorganism, which comprises: 
       a) a disposable bioreactor comprising flexible or semi-flexible waterproof sheets sealed along their edges to form a container, the container having a wedge shaped or rounded bottom at the lowermost extremity of which is positioned a gas bubbler for generating gas bubbles for mixing and providing gases to microbial or cellular liquid cultures within the container, the container further comprising at least one inlet port for introducing gases or liquids and at least one exit port for exhausting gases or removing liquids wherein the exit port further comprises a coiled exhaust tube extending therefrom, and  
       b) instructions for using the disposable bioreactor to culture the microorganisms.  
     
     
       22. The kit of  claim 21 , which further comprises one or more of: 
       c) ingredients for preparing, or prepared culture medium for culturing the microorganism;  
       d) inoculum of the microorganism to be cultured;  
       e) accessories for attaching the bioreactor container to a local source of water for filling the container.  
       f) accessories for connecting the bioreactor inlet port to a gas pump; and  
       g) a gas pump.  
     
     
       23. The kit of  claim 21  wherein the microbial or cellular liquid cultures consist of bacteria, cyanobacteria, fungi, algae, protozoans and nematodes. 
     
     
       24. A disposable bioreactor for culturing microorganisms or cells, comprising flexible or semi-flexible waterproof sheets sealed along their edges to form a container, the container having a generally square or rectangular shape, and further comprising a plurality of gas bubblers positioned across an interior floor of the container, for generating gas bubbles for mixing and providing gases to microbial or cellular liquid cultures within the container, the container further comprising at least one inlet port for introducing gases or liquids and at least one exit port for exhausting gases or removing liquids. 
     
     
       25. The disposable bioreactor of  claim 24 , wherein the length to width ratio of the container is about 1:1 to about 10:1. 
     
     
       26. The disposable bioreactor of  claim 25 , wherein the length to width ratio of the container is between about 2:1 and 5:1. 
     
     
       27. The disposable bioreactor of  claim 24 , wherein the container is two-sided. 
     
     
       28. The disposable bioreactor of  claim 24 , wherein the container is at least three-sided. 
     
     
       29. The disposable bioreactor of  claim 24 , wherein the gas bubbler comprises a gas reservoir comprising inlet holes for admitting gas bubbles from the reservoir to the container interior, the gas reservoir being operably connected to a gas inlet port through which gas is pumped. 
     
     
       30. The disposable bioreactor of  claim 29 , wherein the gas inlet port is connected by a gas inlet tube to a gas pump. 
     
     
       31. The disposable bioreactor of  claim 30 , wherein the gas inlet tube comprises at least one filter for filtering microbial contaminants. 
     
     
       32. The disposable bioreactor of  claim 24 , wherein the exit port further comprises an exhaust tube extending therefrom. 
     
     
       33. The disposable bioreactor of  claim 32 , wherein the exhaust tube is coiled. 
     
     
       34. The disposable bioreactor of  claim 32 , wherein the exhaust tube further comprises a valve to prevent back flushing of liquids to the interior of the container. 
     
     
       35. The disposable bioreactor of  claim 32 , wherein the exhaust tube further comprises a filter for filtering microbial contaminants. 
     
     
       36. The disposable bioreactor of  claim 24 , which further comprises an inoculation port for introducing inoculant into the container. 
     
     
       37. The disposable bioreactor of  claim 36 , wherein the inoculation port comprises a septum. 
     
     
       38. The disposable bioreactor of  claim 24 , which further comprises a breakable pouch on an interior face of the container. 
     
     
       39. The disposable bioreactor of  claim 24 , which further comprises an external reservoir in fluid communication with the container interior. 
     
     
       40. A kit for culturing a microorganism, which comprises: 
       a) a disposable bioreactor for culturing microorganisms or cells, comprising flexible or semi-flexible waterproof sheets sealed along their edges to form a container, the container having a generally square or rectangular shape, and further comprising a plurality of gas bubblers positioned across an interior floor of the container, for generating gas bubbles for mixing and providing gases to microbial or cellular liquid cultures within the container, the container further comprising at least one inlet port for introducing gases or liquids and at least one exit port for exhausting gases or removing liquids; and  
       b) instructions for using the disposable bioreactor to culture the microorganism.  
     
     
       41. The kit of  claim 40 , which further comprises one or more of: 
       c) ingredients for preparing, or prepared culture medium for culturing the microorganism;  
       d) inoculum of the microorganism to be cultured;  
       e) accessories for attaching the bioreactor container to a local source of water for filling the container;  
       f) accessories for connecting the bioreactor inlet port to a gas pump; and  
       g) a gas pump.